Identification of two metallothioneins as novel inhalative coffee allergens cof a 2 and cof a 3.

BACKGROUND: Dust of green coffee beans is known to be a relevant cause for occupational allergic disorders in coffee industry workers. Recently, we described the first coffee allergen (Cof a 1) establishing an allergenic potential of green coffee dust. OBJECTIVE: Our aim was to identify allergenic components of green coffee in order to enhance inhalative coffee allergy diagnosis. METHODS: A Coffea arabica pJuFo cDNA phage display library was created and screened for IgE binding with sera from allergic coffee workers. Two further coffee allergens were identified by sequence analysis, expressed in E. coli, and evaluated by Western blots. The prevalence of sensitization to recombinant Cof a 1, Cof a 2, and Cof a 3 and to commercially available extract was investigated by ELISA (enzyme-linked immunosorbent assay) respectively CAP (capacity test) screening in 18 sera of symptomatic coffee workers. RESULTS: In addition to the previously described chitinase Cof a 1, two Coffea arabica cysteine-rich metallothioneins of 9 and 7 kDa were identified and included in the IUIS Allergen Nomenclature as Cof a 2 and Cof a 3. Serum IgE antibodies to at least one of the recombinant allergens were found in 8 out of 18 symptomatic coffee workers (44%). Only 2 of the analysed sera (11%) had reacted previously to the commercial allergy test. CONCLUSIONS: In addition to the previously described Cof a 1 we have identified two further coffee proteins to be type I coffee allergens (Cof a 2 and Cof a 3) which may have a relevant potential for the specific diagnosis and/or therapy of coffee allergy.

An essential pentatricopeptide repeat protein facilitates 5' maturation and translation initiation of rps3 mRNA in maize mitochondria.

Pentatricopeptide repeat (PPR) proteins are members of one of the largest nucleus-encoded protein families in plants. Here, we describe the previously uncharacterized maize (Zea mays) PPR gene, MPPR6, which was isolated from a Mutator-induced collection of maize kernel mutants by a cDNA-based forward genetic approach. Identification of a second mutant allele and cosegregation analysis confirmed correlation with the mutant phenotype. Histological investigations revealed that the mutation coincides with abnormities in the transfer cell layer, retardation of embryo development, and a considerable reduction of starch level. The function of MPPR6 is conserved across a wide phylogenetic distance as revealed by heterologous complementation of the Arabidopsis thaliana mutant in the orthologous APPR6 gene. MPPR6 appeared to be exclusively present in mitochondria. RNA coimmunoprecipitation and in vitro binding studies revealed a specific physical interaction of MPPR6 with the 5' untranslated region of ribosomal protein S3 (rps3) mRNA. Mapping of transcript termini showed specifically extended rps3 5' ends in the mppr6 mutant. Considerable reduction of mitochondrial translation was observed, indicating loss of RPS3 function. This is consistent with the appearance of truncated RPS3 protein lacking the N terminus in mppr6. Our results suggest that MPPR6 is directly involved in 5' maturation and translation initiation of rps3 mRNA.

Cof a 1: identification, expression and immunoreactivity of the first coffee allergen.

BACKGROUND: Over the past years, dust of green coffee beans has become known to be a relevant cause for occupational type I allergies. Up to now, allergy diagnostics is based on native green coffee bean extract which exhibits insufficient specificity due to interfering substances as well as batch-to-batch variations. No coffee allergen has been described on the molecular level so far. The aim of this study was to identify the first allergen of green coffee. METHODS: The allergenicity of native green coffee bean extracts was analyzed by means of ImmunoCAP in sera of 17 symptomatic coffee workers. A Coffea arabica pJuFo cDNA phage display library was constructed and screened for IgE binding to coffee proteins with 2 sera from allergic coffee workers. By sequence analysis, a new coffee allergen (Cof a 1) was identified, expressed in Escherichia coli, and evaluated by Western blots. The frequency of sensitization was investigated by ELISA screening. RESULTS: The Cof a 1 cDNA encoded a 32-kDa C. arabica class III chitinase. Serum IgE antibodies to the recombinant allergen were found in 3 out of 17 symptomatic coffee workers (18%), whereas only 2 of them reacted to the commercial allergy test. CONCLUSIONS: A class III chitinase of C. arabica was identified to be the first known coffee allergen Cof a 1. It may have a relevant potential for the specific diagnosis of coffee sensitization.

TRANSPARENT LEAF AREA1 encodes a secreted proteolipid required for anther maturation, morphogenesis, and differentiation during leaf development in maize.

We report the identification and functional analysis of TRANSPARENT LEAF AREA1 (TLA1), a maize (Zea mays) gene representing a novel class of secreted, extremely hydrophobic peptides (proteolipids) with a C-terminal Caax box-like motif. ZmTLA1 encodes 27 amino acid residues and is most strongly expressed in the egg cell and microspores. Lower transcript amounts were detected during vegetative development. Transgenic maize expressing an antisense transcript displayed a variety of phenotypes. The most visible phenotypes were dwarfism and transparent leaf areas resulting from defective morphogenesis of mesophyll, bundle sheath, stomatal, and epidermal cells during leaf development. Incomplete cell walls were observed, indicating a defect of cytokinesis. The accumulation of gerontoplasts was probably a secondary effect caused by defects of leaf cell morphogenesis. A defect of anther maturation was observed in approximately 30% of the plants displaying the tla phenotype. Male sterility was mainly caused by incomplete disintegration of the tapetal cell layers and tetrad callose as 90% of the microspores developed into functional pollen. Overexpression of ZmTLA1 seemed to have a lethal effect both in maize and Arabidopsis thaliana. Development of primary roots, root hairs, primary leaves, and chloroplasts was suppressed in Arabidopsis seedlings expressing an inducible ZmTLA1-green fluorescent protein (GFP) fusion protein. GFP signals were exclusively detected in cell walls. Based on our observations, we suggest that the ZmTLA1 peptide represents a class of novel plant morphogens required for the development and maturation of leaf and reproductive tissues.